PROJECT SUMMARY Primary cilia are signaling centers directing sensory signaling in diverse tissues including the brain. Human ciliopathies including Bardet-Biedl syndrome present with a range of deficiencies including retinal degeneration, renal disease, and hyperphagia-driven hypothalamic obesity. Using tandem affinity purification of ciliopathy gene products, the lab has identified key regulators and effectors of these disease genes. Specifically, identification of the BBSome, a complex in central to Bardet-Biedl syndrome provided key insights into the trafficking of GPCRs into cilia. Extending the strategy to look systematically at proteins interacting with the gene products of monogenic obesity syndromes, the Tubby obesity protein was found copurifying with the intraflagellar A complex. Both BBS and Tubby/Tulp3 are important regulators of GPCR trafficking into primary cilia, providing important criteria to identify bona fide ciliary receptors. Using these criteria, the neuropeptide Y family of GPCRs, notably NPY2R, were discovered to be an important determinant of Bardet-Biedl syndrome. This proposal is focused on the newly identified monogenic obesity gene Cep19, which is recruited by FGFR1OP/FOP, CEP350 and a new ciliary GTPase and a candidate obesity gene, Rabl2B. Work here led to the discovery that the IFT-B complex is the central effector of Rabl2B and that Rabl2B is required for IFT-B to assemble cilia. By defining mechanisms regulating the Rabl2B GTPase mechanism, including the identification of GEF and GAP, a critical goal is to understand how the entry of IFT-B into cilia is regulated and how misregulation causes defects in ciliary trafficking of obesity regulators. Additional studies will focus on the ciliary anchors for the Cep19-Rabl2B pathway on the ciliary distal appendage, a set of two pilot studies to look at interactions from two other monogenic obesity genes, adenylate cyclase 3 and Ncs1, and finally to look these trafficking mechanisms controlling GPCR trafficking, the effects of these pathways in Rabl2 knockout mice, and to look for additional human patient lesions in newly identified candidate obesity genes.